Optical analysis of molecules

ABSTRACT

The present invention relates to optical confinements, methods of preparing and methods of using them for analyzing molecules and/or monitoring chemical reactions. The apparatus and methods embodied in the present invention are particularly useful for high-throughout and low-cost single-molecular analysis.

TECHNICAL FIELD

The present invention relates to optical confinements, methods ofpreparing and methods of using them for analyzing molecules and/ormonitoring chemical reactions. The apparatus and methods embodied in thepresent invention are particularly useful for high-throughout andlow-cost single-molecular analysis.

BACKGROUND OF THE INVENTION

Confinement of illumination and signal detection has long beenrecognized as an important tool in molecular diagnostics since theapplication of Fluorescence Correlation Spectroscopy (FCS). FCS involvesillumination of a sample volume containing fluorophore-labeledmolecules, and detection of fluctuations in fluorescence signal producedby the molecules as they diffuse into and out of the effectiveobservation volume. The fluorescence intensity fluctuations can best beanalyzed if the volume under observation contains only a small number offluorescing molecules, and if the background signal is low. This can beaccomplished by the combination of a drastically limited detectionvolume and a low sample concentration. The detection volumes oftraditional FCS are approximately 0.5 femtoliters (or 0.5×10⁻¹⁵ liters),and are achieved through the use of a high numerical aperture microscopeobjective lens to tightly focus a laser beam. In this detection volume,single molecules can be isolated at concentrations of up toapproximately one nanomolar. This concentration range is unacceptablylow for most biochemical reactions, which have reaction constants in themicromolar range. At lower concentrations, these reactions either do notproceed acceptably fast, or behave in a qualitatively different fashion.To observe single molecules at higher concentrations, the observationvolume has to be reduced to far smaller dimensions.

In recent years, the advancement in nanofabrication technology enabledthe production of nanoscale devices that are integrated with electrical,optical, chemical or mechanical elements.

However, there still remains a considerable need for small, massproduced, and disposable devices that can provide optical confinementsof smaller scale, and amenable to single-molecule analysis at a higherconcentration. The present invention satisfies these needs and providesrelated advantages as well.

SUMMARY OF THE INVENTION

A principal aspect of the present invention is the design of opticaldevices and methods for characterizing molecules and/or monitoringchemical reactions. The devices and methods of the present invention areparticularly suited for single-molecule analysis.

Accordingly, the present invention provides an array of opticalconfinements having a surface density exceeding 4×10⁴ confinements permm², wherein individual confinement in the array provides an effectiveobservation volume that is less than one nanoliters (10×⁻⁹ liters),preferably on the order of zeptoliters. In certain aspects, each of theindividual confinement provides an effective observation volume that isless than 100 zeptoliters, or less than 50 zeptoliters, or even lessthan 10 zeptoliters. In other aspects, each of the individualconfinement yields an effective observation volume that permitsresolution of individual molecules present at a concentration that ishigher than one nanomolar, or higher than 100 nanomolar, or on the orderof micromolar range. In certain preferred aspects, each of theindividual confinement yields an effective observation volume thatpermits resolution of individual molecules present at a physiologicallyrelevant concentration, e.g., at a concentration higher than about 1micromolar, or higher than 50 micromolar range or even higher than 100micromolar. The array may comprise zero-mode waveguide or othernanoscale optical structures. The array of optical confinements mayfurther comprise another array of confinements that does not yield theabove-described effective observation volume or does not permitresolution of individual molecules. For example, the array of opticalconfinement can be coupled to a microtiter plate that has a comparablesurface density.

In another embodiment, the present invention provides a method ofcreating a plurality of optical confinements having the aforementionedcharacteristics. The method involve the steps of (a) providing asubstrate; (b) forming an array of optical confinements having a surfacedensity exceeding 4×10⁴ confinements per mm², wherein the individualconfinement comprises a zero-mode waveguide comprising: a claddingsurrounding a core, wherein said cladding is configured to precludepropagation of electromagnetic energy of a wavelength longer than acutoff wavelength longitudinally through the core of the zero-modewaveguide; and (c) illuminating the array with an electromagneticradiation of a frequency less than the cutoff frequency, therebycreating the plurality of optical confinements.

In another embodiment, the present invention provides a method ofcreating an optical observation volume that permits resolution ofindividual molecules. The method involves providing a zero-modewaveguide that comprises a cladding surrounding a core, wherein saidcladding is configured to preclude propagation of electromagnetic energyof a frequency less than a cutoff frequency longitudinally through thecore of the zero-mode waveguide, wherein upon illuminating the zero-modewaveguide with an electromagnetic radiation of a frequency less than thecutoff frequency, the zero-mode waveguide yields an effectiveobservation volume that permits resolution of individual molecules. Incertain aspects, the effective observation volume is less than onenanoliters (10×⁻⁹ liters), preferably on the order of zeptoliters. Usingthe zero-mode waveguide of the present invention, one typically canobtain an effective observation volume that is less than 100 zeptoliters(10×⁻²¹ liters) or less than 50 zeptoliters, or even less than 10zeptoliters. In other aspects, the method yields an effectiveobservation volume that permits resolution of individual moleculespresent at a concentration that is higher than one nanomolar, more oftenhigher than 100 nanomolar, and preferably on the order of micromolarrange. In preferred embodiments, individual molecules present at aconcentration higher than about 5 micromolar, or higher than 7.5micromolar, or even higher than 50 micromolar range, can be resolved bythe method of the present invention.

The present invention also provides a method of detecting interactionsamong a plurality of molecules. The method comprises the steps of (a)placing the plurality of molecules in close proximity to an array ofzero-mode waveguides, wherein individual waveguides in the array areseparated by a distance sufficient to yield detectable intensities ofdiffractive scattering at multiple diffracted orders upon illuminatingthe array with an incident wavelength; (b) illuminating the array ofzero-mode waveguides with an incident wavelength; and (c) detecting achange in the intensities of diffractive scattering of the incidentwavelength at the multiple diffracted orders, thereby detecting theinteractions among a plurality of molecules.

The present invention also provides a method of reducing diffractivescattering upon illuminating an array of optical confinement with anincident wavelength, wherein the array comprises at least a firstoptical confinement and a second confinement, said method comprising:forming the array of optical confinements wherein the first zero-modewaveguide is separated from the second zero-mode waveguide by a distancesuch that upon illumination with the incident wavelength, intensity ofdiffractive scattering resulting from the first zero-mode waveguide at agiven angle is less than that if the first zero-mode waveguide wereilluminated with the same incident wavelength in the absence of thesecond zero-mode waveguide.

Further provided by the present invention is a method of fabricating anarray of optical confinements that exhibits a minimal intensity ofdiffractive scattering of an incident wavelength, comprising: providinga substrate; and forming the array of optical confinements on thesubstrate such that individual confinements in the array are separatedfrom each other at a distance less than one half of the wavelength.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an array of optical confinement, here zero-mode waveguidearranged in a square format. The annotations of various components shownin the figure are as follows; 21: a zero-mode waveguide. 22: “d” is thevariable indicating the inter-zero mode waveguide spacing.

FIG. 2 depicts an array of optical confinement, here zero-modewaveguide, arranged in a non-square format. The annotations of variouscomponents shown in the figure are as follows: 31: the ZMW. 32: thedistance between adjacent ZMWs. 33: the angle formed between the anythree adjacent ZMWs (60 degrees).

FIG. 3 depicts an illustrative 2-dimentional array with an illustrativeangle and two different unit vector lengths. The annotations of variouscomponents shown in the figure are as follows: 41: ZMW. 42: firstdistance d1. 43: unit vector angle. 44: second distance.

FIG. 4 depicts an illustrative regular disposition of ZMWs. In thisconfiguration, there is a lattice defined by the parameters d1, d2 andthe angle 53. In addition to a ZMW at each lattice point, there is acomplex unit cell that comprises a plurality of ZMWs in an arrangementthat is defined by a list of angles and distances with one angle and onedistance for each element of the unit cell. While this figure shows anarray with a unit cell of two components, the unit cell can have anynumber of elements. The annotations of various components shown in thefigure are as follows: 51: ZMW. 52: first lattice distance. 53: latticeangle. 54: second lattice distance. 55: unit cell first distance. 56:unit cell first angle.

FIG. 5 depicts an array of arrays. The annotations of various componentsshown in the figure are as follows: 71: an array of zero modewaveguides. 72: a super array of elements of which the array 71 is aportion thereof.

FIG. 6 depicts the process of negative tone fabrication. The annotationsof various components shown in the figure are as follows: 11: Substrate.12: first resist layer. 13: optional second resist layer. 14: latentchemical modification in the film after exposure. 15: resist featureafter development. 16: deposited metal film. 17: discontinuous metal capdeposited on resist feature. 18: zero mode waveguide structure.

FIG. 7 depicts another illustrative setup. The annotations of variouscomponents shown in the figure are as follows: 81: The ZMW film. 82: theglass coverslip. 83: the integral lenses made of a material with adifferent index of refraction than the glass. 84: the ZMW structure. 85:lines indicating rays of light being focused by the embedded microlens.

FIG. 8, depicts a scanning electron micrographs of ZMW structuresfabricated by positive tone resist (left panels) or negative tone resist(right panels). The grain structure of the polycrystalline film isvisible in the image as flecks, and the ZMWs as dark round structures.In contrast to the irregularly shaped structures that are frequentlygenerated by the positive tone process, the negative tone processconsistently generates highly regular ZMW configurations.

FIG. 9, depicts a single-molecule DNA sequence pattern recognition inZMWs using artificial pre-formed replication forks. (A) Schematics ofthe strand to be synthesized of the preformed fork used here. Only two,asymmetrically spaced R110-dCTPs are to be incorporated in thistemplate. (B) Fluorescence time trace of DNA synthesis from a single DNApolymerase in a ZMW. (C) Histogram of burst intervals from the full timetrace, a section of which is shown in (B).

FIG. 10, depicts an illustrative ZMW coated with a thin film. Theannotations of various components shown in the figure are as follows:101: the ZMW. 102: Side walls inside the ZMW. 103: coating on the uppersurface of the ZMW. 104: metal film. 105: substrate.

FIG. 11 depicts one alignment strategy. The annotations of variouscomponents shown in the figure are as follows: 131: photodetector. 132:optional lens for collecting light. 133: ZMW. 134: metal film. 135:Substrate. 136: objective lens to be aligned. 137: example rays passingthrough objective to ZMW.

FIG. 12 depicts an alternative optical confinement made of porous filmon a substrate. The annotations of various components shown in thefigure are as follows: 91: porous film. 92: pore in porous film. 93:substrate.

FIG. 13 shows an example of a beam mis-aligned on the center of thequadrant detector. The four voltages generated by the four quadrants canbe processed to determine the degree and direction of mis-alignment ofthe beam and thus the ZMW. The annotations of various components shownin the figure are as follows: 111: ZMW. 112: signal generatingmolecules. 113: metal film. 114: substrate. 115: objective lens to bealigned. 116: example rays propagating though system. 117: beamsplitter/dichroic cube. 118: illumination rays incident. 119: returnrays moving toward detector. 120: optional telen lens (used in infinitycorrected systems). 121: more rays. 122: photodetector, possiblyquadrant photodetector.

FIG. 14 depicts an alignment strategy that relies on epi-detectionrather than trans detection. Second image is inset with front view ofquadrant photodiode. The annotations of various components shown in thefigure are as follows: 111: ZMW. 112: signal generating molecules. 113:metal film. 114: substrate. 115: objective lens to be aligned. 116:example rays propagating though system. 117: beam splitter/dichroiccube. 118: illumination rays incident. 119: return rays moving towarddetector. 120: optional telen lens (used in infinity corrected systems).121: more rays. 122: photodetector, possibly quadrant photodetector.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques:

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of Integrated Circuit (IC) processingbiochemistry, chemistry, molecular biology, genomics and recombinantDNA, which are within the skill of the art. See, e.g., Stanley Wolf etal., SILICON PROCESSING FOR THE VLSI ERA, Vols 1-4 (Lattice Press);Michael Quirk et al., SEMICONDUCTOR MANUFACTURING TECHNOLOGY; Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd)edition (1989); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R.Taylor eds. (1995).

Definitions:

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

“Luminescence” is the term commonly used to refer to the emission oflight from a substance for any reason other than a rise in itstemperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when then move from an “excitedstate” to a lower energy state (usually the ground state); this processis often referred to as “radioactive decay”. There are many causes ofexcitation. If exciting cause is a photon, the luminescence process isreferred to as “photoluminescence”. If the exciting cause is anelectron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin-allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin-allowed transitions. Ifphotoluminescence is the result of a spin-forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” or “luminescentsignal” may have any one of the above-described properties.

The term “electromagnetic radiation” refers to electromagnetic waves ofenergy including, in an ascending order of frequency (or alternatively,in a descending order of wavelength), infrared radiation, visible light,ultraviolet (VU) light, X-rays, and gamma rays.

A “primer” is a short polynucleotide, generally with a free 3′-OH group,that binds to a target nucleic acid (or template) potentially present ina sample of interest by hybridizing with the target nucleic acid, andthereafter promoting polymerization of a polynucleotide complementary tothe target.

The terms “operatively linked to” or “operatively coupled to” are usedinterchangeably herein. They refer to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner.

Structure of the Optical Confinements of the Present Invention

One central aspect of the present invention is the design of opticaldevices and methods for characterizing molecules and/or monitoringchemical reactions. Distinguished from the previously reportednanostructures, the optical devices of the present invention allowmultiplexing a massive quantity of single-molecule analysis underphysiologically relevant conditions.

Accordingly, in one embodiment, the present invention provides a highdensity array of optical confinements having a surface density exceeding4×10⁴ confinements per mm², wherein the individual confinement in thearray provides an effective observation volume on the order ofzeptoliters. Preferably, the individual confinement in the arrayprovides an effective observation volume less than about 1000zeptoliters, more preferably less than about 900, more preferably lessthan about 80, even more preferably less than about 10 zeptoliter. Wheredesired, an effective observation volume less than 1 zeptoliter can beprovided. In a preferred aspect, the individual confinement yields aneffective observation volume that permits resolution of individualmolecules present at a physiologically relevant concentration. Thephysiologically relevant concentrations for most biochemical reactionsrange from the micro-molar to millimolar because most of the enzymeshave their Michaelis constants in these ranges. Accordingly, preferredarray of optical confinements has an effective observation volume fordetecting individual molecules present at a concentration higher thanabout 1 micromolar (uM), or more preferably higher than 50 uM, or evenhigher than 100 uM.

To achieve the required observation volume for single-molecule analysesunder physiologically relevant conditions, the subject array generallycomprises zero-mode waveguide or alternative nanoscale opticalstructures. Such alternative structures include but are not limited toporous films with reflective index media, and confinement using indexmatching solids.

As used herein, “zero-mode waveguide” refers to an optical guide inwhich the majority of incident radiation is attenuated, preferably nomore than 80%, more preferably more than 90%, even more preferably morethan 99% of the incident radiation is attenuated. As such high level ofattenuation, no significant propagating modes of electromagneticradiation exist in the guide. Consequently, the rapid decay of incidentelectromagnetic radiation at the entrance of such guide provides anextremely small observation volume effective to detect single-molecules,even when they are present at a concentration as high as in themicromolar range.

The zero-mode waveguide of the present invention typically comprises acladding surrounding a core (i.e., partially or fully), wherein thecladding is configured to preclude propagation of electromagnetic energyof a wavelength higher than the cutoff wavelength longitudinally throughthe core of the zero-mode waveguide. The cladding is typically made ofmaterials that prevent any significant penetration of the electric andthe magnetic fields of an electromagnetic radiation. Suitable materialsfor fabricating the cladding include but not limited to alloys, metals,and semi-conducting materials, and any combination thereof. Alloysinclude any of the numerous substances having metallic properties butcomprising two or more elements of which at lest one is a metal. Alloysmay vary in the content or the amount of the respective elements-whethermetallic or non metallic. Preferred alloys generally improve somedesirable characteristic of the material over a pure elemental material.Characteristics that can be improved through the use of mixtures ofmaterials include, chemical resistance, thermal conductivity, electricalconductivity, reflectivity, grain size, coefficient of thermalexpansion, brittleness, temperature tolerance, conductivity, and/orreduce grain size of the cladding.

In general, alloys suitable for the present invention may involvemixtures where one component is present at fractions as low as 0.0001%.In other instances, alloys with large fractions of more than onecompound will be desirable. One embodiment of the ZMW uses aluminum asthe cladding of the ZMW structure. As an example of how alloys can bebeneficial to a ZMW structure, it is useful to consider different alloysof aluminum in how they would affect a ZMW. In the art of Metalurgy,numerous materials are alloyed with aluminum. Non-limiting examples ofmaterials suitable to alloy with aluminum are antimony, arsenic,beryllium, bismuth, boron, cadmium, calcium, carbon, cerium, chromium,cobalt, copper, gallium, hydrogen, indium, iron, lead, lithium,magnesium, manganese, mercury, molybdenum, nickel, niobium, phosphorous,silicon, vanadium, zinc and others. By way of example of how theintroduction of another element could beneficially impact the ZMWperformance, the introduction of boron to aluminum is known in the artof metallurgy to increase the conductivity of aluminum. An increase inconductivity of the metal film could improve the performance bydecreasing the penetration depth thereby decreasing the observationvolume. A preferred embodiment includes an alloy of aluminum that ismore than 0.0001% of a dopant. A more preferred embodiment includes analloy of aluminum that is more than 0.005% of a dopant. A still morepreferred embodiment includes an allow of aluminum that is more than0.1% of a dopant.

In contrast, some materials are expected to decrease the performance ofthe ZMW structure, and in these instances it will be desirable to takemeasures to eliminate certain impurities. For example, in certainapplications it may be desirable to decrease the amount of lead orarsenic if toxicity of the device is a concern. A preferred embodimentof the device includes a metal film that is less than 1% arsenic. A morepreferred embodiment of the device includes a metal films that is lessthan 0.1% arsenic. A still more preferred embodiment includes a metalfilm that is less than 0.001% arsenic. A still more preferred embodimentincludes a metal film that is less than 0.00001% arsenic. An additionalpreferred embodiment includes a metal film that is less than 1% lead. Astill more preferred embodiment includes a metal film that is less than0.1% lead. A still more preferred embodiment includes a metal film thatis less than 0.01% lead. A still more preferred embodiment includes ametal film that is less than 0.001% lead. A still more preferredembodiment includes a film that is less than 0.00001% lead. In otherapplications where optical confinement performance is especiallyimportant, impurities that tend to reduce the conductivity, therebyworsening the confinement, will be undesirable. For example, vanadium isknown in the art of metallurgy to reduce the conductivity of aluminum. Apreferred embodiment includes a metal film that is less than 0.1%vanadium. A still more preferred embodiment includes a metal film thatis less than 0.01% vanadium. A still more preferred embodiment includesa film that is less than 0.001% vanadium.

Semi-conducting materials suitable for fabricating the cladding aregenerally opaque, and they include silicon, silicates, silicon nitride,silicon dioxide, gallium phosphide, gallium arsenide, or anycombinations thereof.

The cladding of the subject zero-mode waveguide may be coated withmaterials to improve the surface quality. For instance, coating mayenhance the durability of the cladding material. In addition, coating isparticularly desirable if the reactants contained in the core are proneto interact or adhere to the cladding material. A variety of appropriatecoating materials are available in the art. Some of the materials maycovalently adhere to the surface, others may attach to the surface vianon-covalent interactions. Non-limiting examples of coating materialsinclude aluminum oxide film, silanization reagent such asdimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane ortrimethylchlorosilane, polymaleimide, and siliconizing reagents such assilicon oxide, Aquasil™, and Surfasil™

The internal cavity (i.e., the core) surrounded by the cladding mayadopt a convenient size, shape or volume so long as propagating modes ofelectromagnetic radiation in the guide is effectively prevented. Thecore typically has a lateral dimension less than the cutoff wavelength(λ_(c)). For a circular guide of diameter d and having a clad of perfectconductor, λ_(c) is approximately 1.7×d. The cross sectional area of thecore may be circular, elliptical, oval, conical, rectangular,triangular, polyhedral, or in any other shape. Although uniform crosssectional area is preferred, the cross sectional area may vary at anygiven depth of the guide if desired. Preferred average cross sectionalarea ranges from _nm² to _nm².

In a preferred embodiment, the core is non-cylindrical. In one aspect ofthis embodiment, a non-cylindrical core comprises an opening on theupper surface and a base at the bottom surface that is entirely enclosedby the cladding, wherein the opening is narrower in lateral dimensionthan the base. This configuration significantly restricts the diffusionof reactants, and hence increases the average residence time in theobservation volume. Such configuration is particularly useful formeasuring the association rate constant (on-rate) of a chemicalreaction. In another aspect, the core comprises an opening that is widerin lateral dimension than the base. Such configuration allows easieraccess to large molecules that impose a stearic or entropic hinderenceto entering the structure if the open end of the zero mode waveguide wasas small as the base needs to be for optical performance reasons.Examples include the accessibility for long strand polyelectrolytes suchas DNA molecules that are subject to entropic forces opposing entry intosmall openings.

The zero-mode waveguides embodied in the present invention have arelatively high fill fraction ratio, typically above 0.0001, preferablyabove 0.001, more preferably above 0.01, and even more preferably above0.1. As used herein, “fill fraction” of a pattern refers to the ratio ofthe area occupied by the foreground of the pattern to the area occupiedby the background of the pattern. In the context of zero-mode waveguide,the foreground is considered to be the area occupied by the core of thezero-mode waveguide, and the background is the area between thezero-mode waveguide and the cladding (e.g., the aluminum film in certaindesigns). The zero-mode waveguides with high fill fraction ratios areparticularly useful for performing homogenous assays. The fill fractioncan be calculated by summing the total areas of all of the zero-modewaveguides in the array and dividing by the total available areaincluding both the zero-mode waveguides and the spaces between them. Forexample, if a zero-mode waveguide has a diameter of 50 nm, then the areaof this zero-mode waveguide is 7,850 square nanometers. If thesezero-mode waveguides are in a square array separated by 100 nm, thetotal available area is 10,000 square nanometers for each zero-modewaveguide. Therefore, the array has a fill fraction of 78%, which wouldprovide nearly four orders of magnitude higher signal strength in asurface binding assay than a zero-mode waveguide having a fill fractionon the order of 0.01%.

In a bioassay such as an ELISA or other molecular binding bioassay, onelimitation is the inability to operate “homogeneously”, or in a modewhere solutions may be added to a mixture but nothing removed. Thiscomplicates highly multiplexed assays, as provisions for both adding andremoving material from a large number of wells is significantly morecomplex than the provisions for simply adding materials. In the case ofthe ELISA assay, the removal of materials is necessary, because thefluorescent (or other) markers that remain free in solution at the endof the assay would interfere with the ability to detect markers bound tothe reaction surface. Techniques to overcome this have been devised toexploit the short range of radioactive emissions from certainradioisotopes, but these techniques have inherent difficultiesassociated with personnel safety and waste disposal. Other methods forconfining the sensitivity of the assay to the surface have been devised,such as total internal reflection confinement (TIR), and confocaldetection. The zero-mode waveguide photonic structure allows a simplerand less expensive optical system configuration than either of thesetechniques, and vastly outperforms both from the perspective ofconfinement of sensitivity to the surface.

The fill fraction is important in bioassays, because the effective probearea is limited to the surface area of the bottoms of the zero-modewaveguide in the detection region. The amount of signal detectable insuch an assay will be directly proportional to the available area, andhaving a larger fraction of the available surface occupied by zero-modewaveguides will thus increase the signal strength of measurements ofsuch assays. A high fill fraction structure would be generally useful inany surface sensitivity application, not limited to the ELISA assay.

The cutoff wavelength is defined as the wavelength above which thewaveguide is essentially incapable of propagating electromagnetic energyalong the waveguide under the illumination geometry used. Given thegeometry of the core, and the properties of the cladding material, aswell as the wavelength of the incident electromagnetic radiation, oneskilled in the art can readily derive the cutoff wavelength by solvingthe Maxwell's equations (see, e.g., John D. Jackson, CLASSICALELECTRODYNAMICS, second edition, John Willey and Sons). The choice ofthe incident wavelength will depend on the particular application inwhich the subject array is to be employed. In certain aspects, theincident wavelength may be selected from a range of about 10 nm to about1 mm. For detecting fluorescent signals, the incident wavelength istypically selected from the range of about 380 nm to about 800 nm.Polarized (linearly or preferably circularly polarized) or unpolarizedincident radiation is generally employed to illuminate the array inorder to create a desired observation volume.

In a separate embodiment, the present invention provides an alternativeoptical confinement termed external reflection confinement (ERC). Incontrast to the conventional total internal reflection confinement(IRC), the low index medium is the electromagnetic radiation carrier,and the high index (and opaque) medium is the reflector. As such, theroles of the refractive indices are reversed as compared to the IRCsituation. ERC generally requires some kind of means to provide theanalyte (i.e., the molecules under investigation) in the opaque phase.

IRC relies on reflection of an electromagnetic radiation incident on aninterface between high index of refraction and low index of refraction.When light is incident above the critical angle of total internalreflection (known in the art), all of the incident electromagneticradiation is reflected and none is transmitted into the low index phase.A thin region of evanescent radiation is established proximal to theinterface on the low index side. This radiation field is typically anexponentially decaying field with an attenuation length in the rangefrom about 100 nm to about 200 nm, depending on the angle of incidenceand the indices of refraction of the two phases. If the low index phaseis a solution containing an analyte, then the evanescent radiation canbe used to probe the analyte in the solution with a high degree ofsurface sensitivity.

In ERC, the carrier of the propagating electromagnetic radiation is atransparent low index film, and the analyte-bearing medium is ahigh-index metallic opaque film. In this case, most of the radiation isreflected irrespective of the angle of incidence, and non-reflectedlight is rapidly attenuated according to the skin depth of the metal.Typically, means is provided to convey the analyte within the metalphase. Theses means can take the form of a nanocapillary tubeconstructed within the metal layer. When sufficiently small, thepresence of such a tube will have little effect on the distribution ofenergy in the two media, but can be amply large enough to conveybiomolecules. To be small enough, any defects in the metal film must besmall compared with the wavelength of the illumination. This can beachieved because of the large ratio between the wavelength of visiblelight, and the typical size of biomolecules of interest. While visiblelight is typically between 400 nm and 750 nm in wavelength, biomoleculesof interest are generally in the vicinity of 1-30 nm in diameter. Theattenuation of the radiation at the interface can be used to confineillumination to a very small region of the analyte. A small hole in anindex matched (to water) film on a high index substrate could providelateral confinement beyond what is possible with diffraction limitedoptics in the TIR context. This could give 100 zeptoliter confinement inprinciple. In this method, a version of total internal reflectionconfinement is used in which a solid material index-matched to theanalyte solution is applied to the substrate surface and then perforatedwith nanoscale holes. When used in TIR mode, these structures willprovide additional confinement above what can be obtained with TIRalone.

Other alternative confinements are index matching solids. As anillustrative example, such optical confinement can be fabricatedstarting with a high index transparent substrate such as sapphire, spincoat 200 nm of PMMA (polymethyl methacrylate) resist resin. Exposure toelectron beam lithography will render isolated spots soluble accordingto the pattern applied. After development, the device will havenano-scale holes in the PMMA layer and are ready to be used in a TIRsetup. Axial confinement is unaffected by the PMMA layer, as it hasnearly the same index of refraction as the solution containing theanalyte, but the solution is physically prevented from approaching nearthe surface except where the holes are situated, providing a degree oflateral confinement given by the diameter of the holes.

The subject optical confinements can be provided with an optical systemcapable of detecting and/or monitoring interactions between reactants atthe single-molecule level. Such optical system achieves these functionsby first generating and transmitting an incident wavelength to thereactants contained in the confinements, followed by collecting andanalyzing the optical signals from the reactants.

The optical system applicable for the present invention comprises atleast two elements, namely the excitation source and the photondetector. The excitation source generates and transmits incident lightused to optically excite the reactants contained in the opticalconfinement. Depending on the intended application, the source of theincident light can be a laser, a light-emitting diode (LED), aultra-violet light bulb, and/or a white light source. Where desired,more than one source can be employed simultaneously. The use of multiplesources is particularly desirable in case of detecting more than onefluorescent signal to track the interactions of more than one or onetype of molecule simultaneously. A wide variety of photon detectors areavailable in the art. Representative detectors include but are notlimited to optical reader, high-efficiency photon detection system,photodiode (e.g. avalanche photo diodes (APD)), camera, charge coupledevice (CCD), electron-multiplying charge-coupled device (EMCCD),intensified charge coupled device (ICCD), and confocal microscope. Wheredesired, the subject arrays of optical confinement contain variousalignment aides or keys to facilitate a proper spatial placement of theoptical confinement and the excitation sources, the photon detectors, orthe optical transmission element as described below.

The subject optical system may also include an optical transmissionelement whose function is manifold. First, it collects and/or directsthe incident wavelength to the optical confinement containing thereactants. Second, it transmits and/or directs the optical signalsemitted from the reactants inside the optical confinement to the photondetector. Third, it may select and/or modify the optical properties ofthe incident wavelengths or the emitted wavelengths from the reactants.Illustrative examples of such element are diffraction gratings, arrayedwaveguide gratings (AWG), optic fibers, optical switches, mirrors,lenses (including microlens and nanolens), collimators. Other examplesinclude optical attenuators, polarization filters (e.g., dichroicfilter), wavelength filters (low-pass, band-pass, or high-pass),wave-plates, and delay lines. In some embodiments, the opticaltransmission element can be planar waveguides in optical communicationwith the arrayed optical confinements. For instance, a planar waveguidescan be operatively coupled to an array of zero-mode waveguides todirectly channel incident wavelengths to the respective cores of thezero-mode waveguides so as to minimize the loss of wave energy. Theplanar channel can included as a detachable unit located at the base ofarray substrate, or it can be bonded to the substrate as an integralpart of the array.

The optical transmission element suitable for use in the presentinvention encompasses a variety of optical devices that channel lightfrom one location to another. Non-limiting examples of such opticaltransmission devices include optical fibers, diffraction gratings,arrayed waveguide gratings (AWG), optical switches, mirrors, lenses(including microlens and nanolens), collimators, and any other devicesthat guide the transmission of light through proper refractive indicesand geometries.

In a preferred embodiment, the optical confinement of the presentinvention is operatively coupled to a photon detector. For instance, thearrayed optical confinement is operatively coupled to a respective andseparate photon detector. The confinement and the respective detectorcan be spatially aligned (e.g., 1:1 mapping) to permit an efficientcollection of optical signals from the waveguide. A particularlypreferred setup comprises an array of zero-mode waveguides, wherein eachof the individual waveguides is operatively coupled to a respectivemicrolens or a nanolens, preferably spatially aligned to optimize thesignal collection efficiency.

The subject arrays may comprise a single row or a plurality of rows ofoptical confinement on the surface of a substrate, where when aplurality of lanes are present, the number of lanes will usually be atleast 2, more commonly more than 10, and more commonly more than 100.The subject array of optical confinements may align horizontally ordiagonally long the x-axis or the y-axis of the substrate. Theindividual confinements can be arrayed in any format across or over thesurface of the substrate, such as in rows and columns so as to form agrid, or to form a circular, elliptical, oval, conical, rectangular,triangular, or polyhedral pattern. To minimize the nearest-neighbordistance between adjacent optical confinements, a hexagonal array ispreferred.

The array of optical confinements may be incorporated into a structurethat provides for ease of analysis, high throughput, or otheradvantages, such as in a microtiter plate and the like. Such setup isalso referred to herein as an “array of arrays.” For example, thesubject arrays can be incorporated into another array such as microtiterplate wherein each micro well of the plate contains a subject array ofoptical confinements.

As described above, the subject arrays comprise a plurality of opticalconfinements. In some embodiments, the arrays have at least about 20×10⁴distinct optical confinements, preferably at least about 20×10⁶ distinctconfinements, and more preferably at least about 20×10⁸ confinements.The density of the spots on the solid surface in certain embodiments isat least above 4×10⁴ confinements per mm², and usually at least about8×10⁴, at least about 1.2×10⁵, or at least about 4×10⁶ confinements permm², but does not exceed 4×10¹² confinements per mm², and usually doesnot exceed about 4×10¹⁰ confinements per mm². The overall size of thearray generally ranges from a few nanometers to a few millimeters inthickness, and from a few millimeters to 50 centimeters in width orlength. Preferred arrays have an overall size of about few hundredmicrons in thickness and may have any width or length depending on thenumber of optical confinements desired.

The spacing between the individual confinements can be adjusted tosupport the particular application in which the subject array is to beemployed. For instance, if the intended application requires adark-field illumination of the array without or with a low level ofdiffractive scattering of incident wavelength from the opticalconfinements, then the individual confinements should be placed close toeach other relative to the incident wavelength.

Accordingly, the present invention provides array of zero-modewaveguides comprising at least a first and at least a second zero-modewaveguide, wherein the first zero-mode waveguide is separated from thesecond zero-mode waveguide by a distance such that upon illuminationwith an incident wavelength, intensity of diffractive scatteringobserved from the first zero-mode waveguide at a given angle is lessthan that if the first zero-mode waveguide were illuminated with thesame incident wavelength in the absence of the second zero-modewaveguide. A reduced intensity of diffractive scattering is observedwhen the first and the second confinements are placed at the diffractionlimit. Diffractive scattering can be reduced or significantly eliminatedif an array comprises zero-mode waveguides spaced in a regular spacedlattice where the separation of zero-mode waveguides from their nearestneighbors is less than half the wavelength of the incident wavelength.In this regime, the structure behaves as a zero-order grating. Suchgratings are incapable of scattering incident light despite having alarge number of elements that by themselves would scatter veryeffectively. This arrangement is highly desirable for illuminationapproaches such as dark field illumination, where surface scatteringwould cause excitation radiation to be collected by the objective lens,thus increasing background noise. Useful wavelengths for illuminationrange from 250 nm up to 8 microns, meaning that an array of zero-modewaveguides with a spacing of less than 4000 nm would still be useful forapplication in this manner. A spacing of less than 2000 nm is morepreferable, while a spacing of less than 1000 nm is even more preferablein this respect. Some configurations with spacing larger than one halfof the wavelength can have the same advantage if the illumination isapplied asymmetrically, or if the collection cone angle is configured tobe less than 90 degrees. In addition to the benefit of reduceddiffractive scattering, narrow spacing between the individualconfinements decreases the illumination area and thus lowers the powerdemand.

Arrays having the optical confinements spaced far apart relative to theincident wavelength also have desirable properties. While theangle-dependent scattering raises the background signal that could bedisadvantageous for certain applications, it provides a meansparticularly suited for characterizing the size and shape of the opticalconfinements. It also readily permits ensemble bulk measurements ofmolecule interactions, involving especially unlabelled molecules. Arrayssuited for such applications generally contain individual confinementsseparated by more than one wavelength of the incident radiation, usuallymore than 1.5 times the incident wavelength, but usually does not exceed150 times the incident wavelength.

Kits of the Present Invention

The present invention also encompasses kits containing the opticalconfinement arrays of this invention. Kits embodied by this inventioninclude those that allow characterizing molecules and/or monitoringchemical reactions at a single-molecule level. Each kit necessarilycomprises the devices and reagents which render such characterizationand/or monitoring procedure possible. Depending on the intended use ofthe kit, the contents and packaging of the kit will differ. Where thekit is for DNA sequencing, the kit typically comprises: (a) an array ofoptical confinements, preferably zero-mode waveguides of the presentinvention that permits resolution of individual molecules present at aconcentration higher than about 1 micromolar; (b) sequencing reagentstypically including polymerases, aqueous buffers, salts, primers, andnucleoside triphosphates. Where desired, control nucleic acid of knownsequences can be included.

The reagent can be supplied in a solid form or dissolved/suspended in aliquid buffer suitable for inventory storage, and later for exchange oraddition into the reaction medium when the test is performed. Suitableindividual packaging is normally provided. The kit can optionallyprovide additional components that are useful in the procedure. Theseoptional components include, but are not limited to, buffers, capturereagents, developing reagents, labels, reacting surfaces, controlsamples, instructions, and interpretive information. Diagnostic orprognostic procedures using the kits of this invention can be performedby clinical laboratories, experimental laboratories, practitioners, orprivate individuals.

Preparation of the Subject Optical Confinements

The array of the present invention can be manufactured usingnanofabrication techniques provided by the present invention, as well asthose known in the fields of Integrated Circuit (IC) andMicro-Electro-Mechanical System (MEMS). The fabrication processtypically proceeds with selecting an array substrate, followed by usingappropriate IC processing methods and/or MEMS micromachining techniquesto construct and integrate the optical confinement and other associatedcomponents.

Array Substrate:

In some embodiments, the array of optical confinements is present on arigid substrate. In other embodiments concerning, e.g., porous filmswith reflective index media, flexible materials can be employed.

By rigid is meant that the support is solid and does not readily bend,i.e., the support is not flexible. Examples of solid materials which arenot rigid supports with respect to the present invention includemembranes, flexible metal or plastic films, and the like. As such, therigid substrates of the subject arrays are sufficient to providephysical support and structure to optical confinements present thereonor therein under the assay conditions in which the array is employed,particularly under high throughput handling conditions.

The substrates upon which the subject patterns of arrays are may take avariety of configurations ranging from simple to complex, depending onthe intended use of the array. Thus, the substrate could have an overallslide or plate configuration, such as a rectangular or discconfiguration, where an overall rectangular configuration, as found instandard microtiter plates and microscope slides, is preferred.Generally, the thickness of the rigid substrates will be at least about0.01 mm and may be as great as 1 cm or more, but will usually not exceedabout 5 cm. Both the length and the width of rigid substrate will varydepending on the size of the array of optical confinements that are tobe fabricated thereon or therein.

The substrates of the subject arrays may be fabricated from a variety ofmaterials. The materials from which the substrate is fabricated ispreferably transparent to visible and/or UV light. Suitable materialsinclude glass, semiconductors (e.g., silicate, silicon, silicates,silicon nitride, silicon dioxide, quartz, fused silica, and galliumarsenide), plastics, and other organic polymeric materials.

The substrate of the subject arrays comprise at least one surface onwhich a pattern of optical confinements is present, where the surfacemay be smooth or substantially planar, or have irregularities, such asdepressions or elevations. The surface on which the pattern of targetsis presented may be modified with one or more different layers ofcompounds that serve to modulate the properties of the surface in adesirable manner. Modification layers of interest include: inorganic andorganic layers such as metals, metal oxides, polymers, small organicmolecules, functional moieties such as avidin/biotin and the like. Thechoice of methods for applying the coating materials will depend on thetype of coating materials that is used. In general, coating is carriedout by directly applying the materials to the zero-mode waveguidefollowed by washing the excessive unbound coating material. Certaincoating materials can be cross-linked to the surface via extensiveheating, radiation, and by chemical reactions. Those skilled in the artwill know of other suitable means for coating a micro well fabricated onchip, or will be able to ascertain such, without undue experimentation.

Fabrication Process:

Fabrication of the subject chips can be performed according to themethods described as follows or other standard techniques ofIC-processing and/or MEMS micromachining. The standard techniques knownin the art include but are not limited to electron-beam lithography,photolithography, chemical vapor or physical vapor deposition, dry orwet etching, ion implantation, plasma ashing, bonding, andelectroplating. Additional fabrication processes are detailed in theU.S. Patent Application Publication No. 20030174992, the content ofwhich is incorporated by reference in its entirety.

In a preferred embodiment, the present invention provides a negativetone fabrication process. Unlike the conventional positive tonefabrication process that suffers from the drawback of creating opticalconfinements of varying dimensions, the subject negative tone processprovides far more consistent configurations. A comparison of the twofabrication processes is shown in Table 1 below. TABLE 1 Positive andNegative Tone Process Steps in Fabrication of Zero-Mode Waveguides Step# Positive Tone Process Negative Tone Process 1 Clean fused silicasubstrates Same in heated solution of hydrogen peroxide and ammoniumhydroxide. 2 Cascade rinse substrates in Same deionized water. 3 Cleansubstrates in oxygen Same plasma cleaner. 4 Coat substrates with metalSpin-coat substrates with film by either thermal electron-beam resist.evaporation or sputtering. 5 Spin-coat substrates with Bake castingsolvent out electron-beam resist over the of film. metal layer. 6 Bakecasting solvent out of Expose resist with electron film. beamlithography. 7 Expose resist with electron Develop resist in chemicalbeam lithography. bath to reveal array of small pillars with large emptygaps in resist. 8 Develop resist in chemical Rinse developer away andbath to reveal holes. dry chips. 9 Rinse developer away and dry Coatchips with metal film chips. by either thermal evaporation orsputtering. 10 Use reactive-ion etching to Dissolving underlyingtransfer resist pattern into negative resist using metal film.Microposit 1165 Stripper. 11 Strip resist using oxygen Same plasma.

In a negative tone process, a negative resist is applied to thesubstrate. A resist is negative if it is rendered insoluble byapplication of some agent, where in some cases the agent is opticalenergy or electron beam energy. Alternatively, a positive tone resistcan be used with a negative pattern. A negative tone pattern ischaracterized by the application of the agent in all areas except thelocation of the optical confinement, e.g., zero-mode waveguide,contrasted with a positive tone image in which the agent is confinedonly to the optical confinement area. In either case, after developmentof the resist, resist remains only in the areas where the opticalconfinement is intended to lie. It is useful in many cases to use meansto achieve an undercut sidewall profile of these remaining resistfeatures. Many techniques exist in the art to obtain undercut sidewalls,for example, in electron beam lithography. For instance, one method isto apply to layers of electron beam resist to the surface sequentially,the upper film having a higher sensitivity to the energy delivered to itby the electron beam. Because the beam has a tendency to spread, alarger area of the upper film will be rendered insoluble than in thelower layer, resulting in an overhang beneath the upper layer asdesired.

After development and appropriate cleaning procedures known in the artsuch as a plasma de-scum procedure, the metal film comprising theoptical confinement can be applied by one of several methods, includingmetal evaporation, molecular beam epitaxy and others. In the case thatthe resist profile is undercut as discussed above, the metal that isdeposited in the regions still occupied by the resist will rest on topof the resist rather than resting on the device surface. The resistlayer is subsequently removed by any of several techniques includingsolvent dissolution either with or without ultrasonication or othermechanical agitation, reactive plasma etching, vaporization or others.The metal which rested on the resist features is removed as the resistis removed (“lifted off”), while the resist resting directly on thesubstrate remains to form the walls of the optical confinement.

The advantage of this process is that the size of the opticalconfinement is determined by the size of the resist feature, and doesnot rely on the fidelity of reactive ion etch pattern transfermechanisms, which can be highly variable for metal films, especiallyAluminum a desirable metal for these devices. The positive tone processis subject to the inherent variation in resist feature sizes plus thevariation due to pattern transfer, while the negative tone process issubject to the first variability but not the second. Metal thin filmtechniques suffer from much less lateral variation, and so the overallaccuracy is better. This method also does not rely on the availabilityof a suitable etch for the metal in question, allowing the applicationof the process to a much wider selection of metals than the positivetone process.

FIG. 6 is a schematic presentation of an illustrative negative toneprocess to make zero-mode waveguide. In this process, the substrate 11is first coated with a layer of negative resist 12. Optionally, thesubstrate can be coated with a second resist layer 13. Exposure of theresist to the same pattern electron beam lithography tool used in thepositive tone process, generates the opposite pattern as previouslyobserved, namely one of a periodic array of small pillars of remainingresist, and empty gaps between the pillars 15. The final zero-modewaveguide structures are created by coating this pattern with a thinmetal layer such as an aluminum layer 17, and then dissolving theunderlying negative resist pillars 18. Because this process is notdependent on the thickness of the alumina layer or the crystal structureor morphology of the metal film, it produces a far more consistentconfiguration, and provides much finer control over the critical featuresize.

A variant negative tone process is termed nanocasting. The steps ofnanocasting are similar except that the use of bi-layer resist isavoided. The process first involves depositing on the surface of asubstrate (in this case a single-layer resist would be used). Theelectron beam exposure and development follow, leaving a cylindricalfeature for each dot in the exposure pattern. For this process, it isdesirable to allow the metal deposition technique to apply material notjust on the top of the resist structure but also on the sidewalls of theresist feature. This process is inherently three dimensional, in that anegative replica of the exterior surface of the three-dimensional resistfeature is reproduced in the interior surface of the metal films thatforms the optical confinement walls. In this case, the undercut resistprofile and the various methods used to produce this are not necessary,as in the negative tone process, they are use specifically to preventcontact of the deposited film with the sides of the resist feature. Inthe nanocasting approach, the deposited film faithfully reproduces theexterior surface of the resist feature, so an undercut figure would onlybe used if a non cylindrical confinement is desired.

In practicing nanocasting, cautions must be employed to removed themetal from above the nanocasting “master” (the resist feature), as theresist feature can in some instances be entirely buried and unavailablefor removal. This, however, can be remedied in a number of ways.

Where the deposition technique has a high degree of anisotropy in thedeposition (such as metal evaporation), the sidewalls will be very thinnear the top of the resist feature, which in some instances can be acylindrical pillar. This weak point can be subject to direct mechanicaldisruption allowing the removal of the metal above the resist featureand hence the ZMW location. An isotropic etch, either solution phase orplasma can be used to further thin the film until this weak pointseparates, achieving the same effect. If the metal deposition step has alow degree of anisotropy (such as sputtering or electroplating), thenthe resist material can be exposed through chemical mechanicalpolishing, or ion milling.

Simultaneous with or subsequent to the removal of the metal cap over theresist feature, the resist material is then removed by solventdissolution, or reactive ion etching. This completes the fabricationsteps, provided the appropriate pattern is applied and the otherparameters are correctly chosen.

Uses of the Subject Optical Confinements and Other Devices of thePresent Invention

The subject devices including optical confinements and associatedoptical systems provide an effective means for analyzing molecules andreal-time monitoring chemical reactions.

In certain aspects, the subject devices and methods provideunprecedented performance in single-molecule observation. First, itprovides information on individual molecules whose properties are hiddenin the statistical mean that is recorded by ordinary ensemblemeasurement techniques. In addition, because it can be multiplexed, itis conducive to high-throughput implementation, requires smaller amountsof reagent(s), and takes advantage of the high bandwidth of modernavalanche photodiodes for extremely rapid data collection. Moreover,because single-molecule counting automatically generates a degree ofimmunity to illumination and light collection fluctuations,single-molecule analysis can provide greater accuracy in measuringquantities of material than bulk fluorescence or light-scatteringtechniques. As such, the subject device and detection/monitoring methodsmay be used in a wide variety of circumstances including sequencingindividual human genomes as part of preventive medicine, rapidhypothesis testing for genotype-phenotype associations, in vitro and insitu gene-expression profiling at all stages in the development of amulti-cellular organism, determining comprehensive mutation sets forindividual clones and profiling in various diseases or disease stages.Other applications involve profiling of cell receptor diversity,identifying known and new pathogens, exploring diversity towardsagricultural, environmental and therapeutic goals.

In a preferred aspect, the subject devices including various forms ofoptical confinements and the associated optical systems are particularlysuited for conducting single-molecule DNA sequencing. Procedures forconducting single-molecule sequencing are fully described in U.S. Ser.No. 09/572,530, the content of which is incorporated by reference in itsentirety.

EXAMPLE 1

The following provides an illustrative process of fabricating zero-modewaveguide. The parameters described herein are meant to be illustrativeand not intended to be limiting in any manner.

-   -   1. Substrates: Substrates are double polished, 60/40 scratch/dig        surface quality, Fused Silica wafers, cut to 100        millimeters(+/−0.2 mm) diameter, and 175 micrometer(+/−25        micrometers) thick and a total thickness variation of less than        25 micrometers.    -   2. Clean: A mix of 5 parts deionized water, 1 part of (30% v/v        Hydrogen Peroxide in water), 1 part of (30% v/v Ammonium        Hydroxide in water) is heated to 75 degree Celsius on a        hotplate. The wafers are immersed in the mix using a Teflon        holder for a duration of 15 minutes.    -   3. Rinsing: The holder containing the wafers is removed from the        RCA clean bath and immersed in a bath of deionized water. The        wafers are left in this second bath for a 2 minutes period. The        holder still containing the wafers is removed from the bath, and        sprayed with deionized water to thoroughly finish the rinsing        process.    -   4 Drying: Within a minute of the final rinsing step, the wafers        are dried, while still in the holder, using a dry clean nitrogen        flow.    -   5. Oxygen Plasma: The wafers are then placed in a Glenn 1000p        plasma Asher, used in plasma etch mode (wafers on a powered        shelf, and under another powered shelf), with 140 mTorr pressure        and 400 Watts of forward power at 40 kHz frequency. The plasma        is maintained for 10 minutes. A flow of 18 sccm of molecular        oxygen is used.    -   6. Vapor Priming: The wafers are loaded within 3 minutes after        the Oxygen plasma in a Yield Engineering Systems vapor priming        oven where they are coated with a layer of HexaMethylDiSilazane        (HMDS) adhesion promoter.    -   7. Electron beam resist coating: The wafers are coated within 15        minutes after the Vapor Priming in a manual spinner unit using        NEB-31 electron beam resist (Sumitomo Chemical America). About 3        ml are dispensed on the wafer, which is then spun at 4500 rpm        for 60 seconds. Initial acceleration and deceleration are set to        3 seconds    -   8. Resist Bake: The wafers are baked on a CEE hotplate at a        temperature of 115 degree Celsius for 2 minutes. The plate is        equipped with a vacuum mechanism that allows good thermal        contact between the wafers and the hotplate surface.    -   9. Gold Evaporation: a layer of 10 nm of gold is then thermally        evaporated on the Wafers, on the side coated with the resist. A        pressure of less than 2 10e-06 Torr must be reached before the        evaporation. The evaporation is performed at a rate of        approximately 2.5 Angstrom per second and monitored using an        Inficon controller.    -   10. Electron beam exposure: a pattern consisting of Zero Mode        Waveguides is exposed on the wafers, using a high resolution        electron beam lithography tool such as a Leica VB6-HR system.        Zero mode waveguides are patterned as single exel features. At a        current of nominally 1 nanoAmpere, and a Variable Resolution        Unit of 1, and for an exel setting of 5 nanometers, doses can        range from 10000 microCoulombs per square centimeters to 300000        microCoulombs per square centimeters are used.    -   11. Gold Etch: After removal from the electron beam system, the        10 nanometer gold layer is removed using gold etchant TFA at        room temperature (GE 8148, Transene Corporation), for 10        seconds. Wafers are held in a Teflon holder similar to the one        used in step 2.    -   12. Rinsing: The holder containing the wafers is removed from        the gold etchant bath and immerse in a bath of deionized water.        The wafers are left in this second bath for a 2 minutes period        with gentle manual agitation. The holder still containing the        wafers is removed from the bath, and sprayed with deionized        water to thoroughly finish the rinsing process.    -   13. Drying: Within a minute of the final rinsing step, the        wafers are dried, While still in the holder, using dry clean        nitrogen flow.    -   14. Post Exposure Bake: The wafers are then submitted to a 2        minute post exposure bake on a hotplate at 95 degree Celsius,        equally equipped with a vacuum mechanism.    -   15. Developing: The wafers are loaded in a Teflon holder and        immersed in developer MF-321 (Shipley Chemicals, Rohm-Haas) at        room temperature for duration of 30 seconds. Wafers are held in        a Teflon holder similar to the one used in step 2.    -   16. Rinsing: The holder containing the wafers is removed from        the developer etchant bath and immerse in a bath of deionized        water. The wafers are left in this second bath for a 2 minutes        period with gentle manual agitation. The holder still containing        the wafers is removed from the bath, and sprayed with deionized        water to thoroughly finish the rinsing process.    -   17. Drying: Within a minute of the final rinsing step, the        wafers are dried, while still in the holder, using dry clean        nitrogen flow.    -   18. Surface Descum: The wafers are loaded in a Glenn 1000p        plasma asher run in ashing mode (Wafers on a grounded plate        below a powered plate), and submitted to a 30 seconds surface        descuming oxygen plasma at a pressure of 140 mTorr and a power        of 100 Watts forward power at 40 kHz. A flow of 18 sccm of        molecular oxygen is used.    -   19. Aluminium Evaporation: The wafers are loading in a metal        evaporator within 5 minutes of the surface descum process. A        layer of 100 nm of thermally evaporated Aluminium is now        deposited on the wafers. Evaporation is made at a pressure of no        less than 2 10ˆ-6 Torr at a rate of 25 Angstrom per seconds and        monitored using an Inficon controller.    -   20. Aluminium Thickness measurement: The thickness of the        aluminium is measured using a P-10 Profilometer (Tencor).    -   21. Zero Mode Waveguide Decasting: The Zero Mode Waveguide are        decasted from the enclosing Aluminium film by immersing them, in        a Teflon holder, in a bath of 1165 Stripper (Shipley Chemicals,        Rohm-Haas), or in a bath of AZ-300T Stripper (Shipley Chemicals,        Rohm-Haas). The bath is submitted to sonication by immersing the        Container holding both the Stripper and the wafer holder in a        sonicator. The wafers are left in the decasting bath for 30        minutes    -   22. Rinsing: The stripping bath is removed from the sonicator.        The holder containing the wafers is removed from the stripper        bath and immerse in a bath of deionized water. The wafers are        left in this second bath for a 2 minutes period with gentle        manual agitation. The holder still containing the wafers is        removed from the bath, and sprayed with deionized water to        thoroughly finish the rinsing process.    -   23. Drying: Within a minute of the final rinsing step, the        wafers are dried, while still in the holder, using dry clean        nitrogen flow    -   24. Photoresist coating: The wafers are coated with Shipley 1827        photoresist spun at a speed of 1500 rpm. About 5 ml of resist is        dispensed. Acceleration and deceleration is set to 5 seconds.    -   25. Resist Bake: The wafers are baked on a CEE hotplate at a        temperature of 115 degree Celsius for 15 minutes. The plate is        equipped with a vacuum mechanism that allows good thermal        contact between the wafers and the hotplate surface.    -   26. Dicing: The wafer are diced using a K&S-7100 dicing saw        (Kulicke & Soffa) using a resin/diamond blade (ADT        00777-1030-010-QIP 600). The wafers are mounted on a low-tack        adhesive tape prior to dicing.    -   27. Die Removal: The dies are removed from the adhesive tape        manually and stored.    -   28. Resist removal: The layer of 1827 photoresist is removed by        immersing the dies first in an acetone bath for 1 minute, then        in a 2-propanol bath for 2 minute with gentle manual agitation.    -   29. Die Drying: The die is dried after being removed from the        2-propanol bath using dry clean air.    -   30. Plasma Clean: The wafers are loaded in a Drytek 100 plasma        etcher, and submitted to a 1 minute oxygen plasma at a pressure        of 140 mTorr, a molecular oxygen flow of 85 sccm oxygen and an        RF power of 500 Watts forward power at 13 Mhz.

EXAMPLE 2

Monitoring Enzymatic Synthesis of a DNA Strand by a Single DNAPolymerase Molecule in Real Time

In these experiments we sought to track the enzymatic synthesis of a DNAstrand by a single DNA polymerase molecule in real time using afluorescently labeled nucleotide. We immobilized individual Phi29^(N62D)DNA polymerase enzymes (Amersham Biosciences, Piscataway, N.J.) inzero-mode waveguides (ZMWs) by non-specific binding using a diluteenzyme solution. After immobilization, the ZMW structures were washed toremove unbound enzyme, and then exposed to a solution containing thereaction reagents. As for the DNA template, we used a 68-bp pre-primedcircular DNA that contained two cytosine bases in characteristic,asymmetric spacing (FIG. 9A). Strand-displacement polymerizing enzymessuch as Phi29 DNA polymerase will continuously loop around the circulartemplate and thus generate a long and highly repetitive complementaryDNA strand.

We used R110-dCTP (Amersham Biosciences, Piscataway, N.J.) as thefluorescently-tagged nucleotide analog in which the fluorophore isattached to the nucleotide via a linker to the gamma-phosphate. Incontrast to the more commonly used base-labeled nucleotide analogs,gamma-phosphate-linked analogs are cleaved through the enzymaticactivity of DNA polymerase as the attached nucleotide is incorporatedinto the growing DNA strand and the label is then free to diffuse out ofthe effective observation volume surrounding the DNA polymerase. Theefficient removal of the fluorophore ensures continuously low backgroundlevels and prevents significant interference with DNA polymeraseactivity. These features of the gamma-phosphate-linked fluorophore arepreferable for this application because they will enable replacement ofall four bases with fluorophore-tagged analogs, as is generally requiredfor full implementation of the DNA sequencing application. We candistinguish binding of a nucleotide and its subsequent incorporationinto nucleic acid from a mismatch event because the rate constants ofthese two processes are significantly different, and because nucleotideincorporation involves several successive steps that prevent zero delaytime events.

All other nucleotides were supplied without labels. We have establisheda very effective way of removing any remaining trace amount of nativedNTP in a nucleotide analog preparation to ensure that errors are notintroduced due to the incorporation of unlabeled dNTPs by an enzymaticpurification using an alkaline phosphatase prior to the polymerizationassay.

To investigate the speed and processivity of the Phi29^(N62D) DNApolymerase under these conditions, we measured incorporationcharacteristics using R110-dCTP completely replacing dCTP in thereaction mixture, both in solution and with enzyme immobilized on aglass surface. We found that the enzyme efficiently utilizes thisanalog, synthesizing complementary DNA of many thousands of base pairsin length without interruption in a rolling circle synthesis protocol,using both small preformed replication forks (FIG. 9A) as well as largercircular DNA such as M13 DNA. Similar experiments demonstrated that DNApolymerase can be immobilized to the bottom of ZMWs without loosing thiscatalytic activity.

We tracked the incorporation of the fluorescently labeled dCTPnucleotide during rolling-circle DNA synthesis by recording thefluorescent light bursts emitted in an individual ZMW. DNA polymeraseactivity was observed in many waveguides as distinct bursts offluorescence, lasting for several minutes. The fluorescence time traceshowed a characteristic double burst pattern (FIG. 9B), each burstcorresponding to an incorporation event of a R110-dCTP analog into theDNA strand and subsequent cleavage of the fluorophore. In histograms ofburst intervals derived from the full time trace, two peakscorresponding to DNA synthesis along the short (14 bases, approximatelyone second) and long (54 bases, approximately four seconds) DNA templatesegments are visible, consistent with an overall average speed measuredin bulk solution under these conditions of approximately ten base pairsper second.

It is noteworthy that we could observe this single-molecule activity ata fluorophore concentration of 10 μM. In conventionally createdexcitation volumes, the number of fluorophores would be far too high topermit the observation of individual enzymatic turnovers of DNApolymerase. These experiments thus confirmed the validity of theZMW-based single-molecule DNA sequencing approach by verifying that (a)immobilization of DNA polymerase in ZMWs does not affect its enzymaticactivity; (b) fluorescent gamma-phosphate-linked nucleotide analogs donot inhibit the activity of DNA polymerase; and (c) ZMWs provide anadequate degree of confinement to detect single-molecule DNA polymeraseactivity at physiological concentrations of reagents. More generally,these results prove that ZMWs allow single-molecule analysis of enzymekinetics, especially involving any enzyme that can be attached to thesurface and for which substrates can be fluorescently labeled.

1. An array of optical confinements having a surface density exceeding4×10⁴ confinements per mm², wherein individual confinement in the arraypermits resolution of individual molecules present at a concentrationhigher than about 1 micromolar.
 1. An array of optical confinementshaving a surface density exceeding 4×10⁴ confinements per mm², whereinindividual confinement in the array provides an effective observationvolume less than about 1000 zeptoliters.
 2. A method of detectinginteractions among a plurality of molecules, comprising: placing theplurality of molecules in close proximity to an array of zero-modewaveguides, wherein individual waveguides in the array are separated bya distance sufficient to yield detectable intensities of diffractivescattering at multiple diffracted orders upon illuminating the arraywith an incident wavelength; illuminating the array of zero-modewaveguides with an incident wavelength; and detecting a change in theintensities of diffractive scattering of the incident wavelength at themultiple diffracted orders, thereby detecting the interactions among aplurality of molecules.
 3. A method of reducing diffractive scatteringupon illuminating an array of optical confinement with an incidentwavelength, wherein the array comprises at least a first opticalconfinement and a second confinement, said method comprising: formingthe array of optical confinements wherein the first zero-mode waveguideis separated from the second zero-mode waveguide by a distance such thatupon illumination with the incident wavelength, intensity of diffractivescattering resulting from the first zero-mode waveguide at a given angleis less than that if the first zero-mode waveguide were illuminated withthe same incident wavelength in the absence of the second zero-modewaveguide.
 4. A method of fabricating an array of optical confinementsthat exhibits a minimal intensity of diffractive scattering of anincident wavelength, comprising: providing a substrate; and forming thearray of optical confinements on the substrate such that individualconfinements in the array are separated from each other at a distanceless than one half of the wavelength.
 5. A method of creating aplurality of optical confinements having a surface density exceeding4×10⁴ confinements per mm², wherein individual confinement in the arraypermits resolution of individual molecules present at a concentrationhigher than at least about 1 micromolar, comprising: providing asubstrate; forming an array of optical confinements having a surfacedensity exceeding 4×10⁴ confinements per mm², wherein the individualconfinement comprises a zero-mode waveguide comprising: a claddingsurrounding a core, wherein said cladding is configured to precludepropagation of electromagnetic energy of a frequency less than a cutofffrequency longitudinally through the core of the zero-mode waveguide;and illuminating the array with an electromagnetic radiation of afrequency less than the cutoff frequency, thereby creating a pluralityof optical confinements.